Presently there are two basic approaches to DNA sequence determination: the chain termination method, e.g. Sanger et al, Proc. Natl. Acad. Sci., 74: 5463-5467 (1977); and the chemical degradation method, e.g. Maxam et al, Proc. Natl. Acad. Sci., 74: 560-564 (1977). The chain termination method has been improved in many ways since its invention, and serves as the basis for all currently available automated DNA sequencing machines, e.g. Sanger et al, J. Mol. Biol., 143: 161-178 (1980); Schreier et al, J. Mol. Biol., 129: 169-172 (1979); Smith et al, Nature, 321: 674-679 (1987); Prober et al, Science, 238: 336-341 (1987); Hunkapiller et al, Science, 254: 59-67 (1991); Bevan et al, PCR Methods and Applications, 1: 222-228 (1992). Moreover, further improvements are easily envisioned that should greatly enhance the throughput and efficiency of the approach, e.g. Huang et al, Anal. Chem., 64: 2149-2154 (1992)(capillary arrays); Best et al, Anal. Chem., 66: 4063-4067 (1994)(non-cross-linked polymeric separation media for capillaries); better dye sets; and the like.
Nonetheless, even with such reasonably envisioned improvements, these approaches still have several inherent technical problems that make them both expensive and time consuming, particularly when applied to large-scale sequencing projects. Such problems include i) the gel electrophoretic separation step which is labor intensive, is difficult to automate, and which introduces an extra degree of variability in the analysis of data, e.g. band broadening due to temperature effects, compressions due to secondary structure in the DNA sequencing fragments, inhomogeneities in the separation gel, and the like; ii) nucleic acid polymerases whose properties, such as processivity, fidelity, rate of polymerization, rate of incorporation of chain terminators, and the like, are often sequence dependent; iii) detection and analysis of DNA sequencing fragments which are typically present in fmol quantities in spacially overlapping bands in a gel; iv) lower signals because the labelling moiety is distributed over the many hundred spacially separated bands rather than being concentrated in a single homogeneous phase, v) in the case of single-lane fluorescence detection, the availability of dyes with suitable emission and absorption properties, quantum yield, and spectral resolvability; and vi) the need for a separately prepared sequencing template for each sequencing reaction to identify a maximum of about 400-600 bases, e.g. Trainor, Anal. Biochem., 62: 418-426 (1990); Connell et al, Biotechniques, 5: 342-348 (1987); Karger et al, Nucleic Acids Research, 19: 4955-4962 (1991); Fung et al U.S. Pat. No. 4,855,225; Nishikawa et al, Electrophoresis, 12: 623-631 (1991); and Hunkapiller et al (cited above).
The need to prepare separate sequencing templates is especially onerous in large-scale sequencing projects, e.g. Hunkapiller et al (cited above)(94.4 kilobase target--2399 templates); and Alderton et at, Anal. Biochem., 201: 166-169 (1992)(230 kilobase target--13,000 templates).
Attempts to automate template preparation have proved difficult, especially when coupled with current sequencing methodolgies, e.g. Church et al, Science, 240: 185-188 (1988); Beck et al, Anal. Biochem. 212: 498-505 (1993); Wilson et al, Biotechniques, 6: 776-787 (1988); and the like.
In view of the above, a major advance in sequencing technology would take place if there were means available for overcoming the template-preparation bottleneck. In particular, the ability to prepare many thousands of templates simulaneously without individual template selection and handling would lead to significant increases in sequencing throughput and significant lowering of sequencing costs.